Synthesis of 1, 2-dehydro steroid by cylindrocarpon radicicola or fusarium javanicum



United States Patent SYNTHESIS OF 1,2-DEHYDRO STEROID BY CYLIN-' DROCARPON RADI'CICOLA JAVANICUM 8 Claims. (Cl. 195-51) OR FUSARIUM This application is a continuation-in-part of our parent application, Serial No. 331,333, filed January 14, 1953, now Patent No. 2,744,120, granted May 1, 1956.

This invention relates to the microbiological conversion of steroids, and has for its object the provision of a process for converting Reichsteins Compound S or a 21-ester thereof to a 1,2-dehydro derivative (i. e. A pregnadien-17a,21-diol-3,20-dione or a 21-ester thereof) by means of certain microorganisms. [Reichsteins Compound S, which is n -pregnene-17a,21-diol-3,20-dione, will be referred to for brevity as Compound S or S.]

More specifically, this invention essentially comprises subjecting Compound S or a 21-ester thereof to the action of fungi (or enzymes thereof) of the order Moniliales, family Tuberculariaceae, preferably of the genera Cylindrocarpon and Fusarium, and recovering the 1,2-de-hydro steroid thus formed. Thus, it has been found that the fungi Cylindrocarpon radicicola ATCC 11011, and Fusarium javanicum var. ensiforme QM 524 [Quartermaster Culture Collection, Quartermaster General Laboratories, Philadelphia, Pennsylvania] have a high order of activity in the microbiological dehydrogenation of this invention, each of these organisms forming, inter alia, the known steroid 1,2-dehydro-S, which has known value as an intermediate in the preparation of 1,2-dehydrohydrocortisone and other physiologically-active steroid derivaties. The organisms also form, under certain conditions, A -testololactone and A -pregnadiene-17a,20fl,21-triol- 3-one, the latter beinga new steroid which can be oxidized after 21-monacylation to a 21-ester of Fusarium javanicum var. ensiforme has also been deposited with the American Type Culture Collection, Washington, D. C., wherein it has been assigned catalogue No. 12575.

In the practice of this invention, the dehydrogenation may be effected in a growing culture by either adding Compound S or a 21-ester thereof to the culture during the incubation period, or by including it in the nutrient medium prior to inoculation. In any case, assimilable sources of nitrogenous materials (for growth-promotion) and carbon-containing materials (as energy source) should be present in the culture medium. Also, an adequate, sterile air supply should be maintained during the oxidation, e. g. by the conventional techniques of (1) exposing a large surface of the medium to air, or (2) submerged culture.

As the steroid substrate, either Compound S itself or a 2l-ester thereof may be used. Suitable esters include the aliphatic acid esters (e. g. the alkanoic acid esters, and especially the lower alkanoic acid esters as exemplified by the acetate, propionate, butyrate, and enanthate), the aromatic acid esters (e. g. the hydrocarbon-aromatic acid esters as exemplified by the benzoate), the araliphatic acid esters (e. g. the hydrocarbon aralkanoic acid esters as exemplified by phenylacetate and B-phenylpropi'onate). The particularly preferred esters are those of hydrocarbon carboxylic acids having less than ten carbon atoms.

The sources of nitrogenous, growth-promoting factors are those normally employed in such processes. They may be natural organics (e. g. soybean meal, cornsteep liquor, meat extract and/or distillers solubles) or synthetics such as nitrates and ammonium compounds.

- Suitable energy-source material which may be utilized in the process of this invention includes (a) lipids, especially (1) fatty acids having at least 14 carbon atoms, (2) fats, or (3) mixtures thereof, as exemplified by such fats as lard oil, soybean oil, linseed oil, cottonseed oil, peanut oil, coconut oil, corn oil, castor oil, sesame oil, crude palm oil, fancy mutton tallow, sperm oil, olive oil, tristearin, tripalmitin, triolein, and trilaurin; and such fatty acids as stearic, palmitic, oleic, linoleic and myristic acids; and (b) other carbon-containing materials as exemplified by gylcerol, glucose, fructose, dextrose, sucrose, lactrose, maltose, dextrins, starches and whey. These materials may be used either in purified state or as concentrates, such as whey concentrate, cornsteep liquor, or grain mashes (e. g. corn, wheat or barley mash). Mixtures of the above may, of course, be employed. It is to be noted, however, that the steroid substrate isadded to the fermentation medium essentially as a precursor, and not as an energy source.

The following examples are illustrative of the invention, but are not to be construed as a limitation thereof.

Example I (a) Fermentati0n.A medium of the following composition is prepared: corn steep liquor, 6.0 g.; NH4H PO 3.0 g.; CaCO 2.5 g.; soybean oil, 2.2 g.; yeast extract, 2.5 g.; dextrose, 10.0 g.; and distilled water to make 1 liter. The medium is adjusted to pH 7.0i0.1. Then, 50 ml. portions of the medium are distributed in 250 ml. Erlenmeyer flasks, and the flasks plugged with cotton and sterilized in the usual manner (i. e. by autoclaving for 30 minutes at 120 C.). When cool, each of the flasks is inoculated with a suitable spore suspension of Cylindrocarpon redicicola AT CC 11011, e. g., about one-fifth of the suspendable surface-growth from a 7 to 10 day old Sabouraud agar slant. After hours incubation at 25 C. on a 280 cycles per minute rotary shaker, 10% by volume transfer is made to a fresh medium of the above composition. Incubation is continued for 48 hours; then 1.013 g. of Compound S in 40.5 ml. of absolute methanol is added in the amount of 0.5 ml. per flask. After an additional 47 hour period the cultures are filtered through a Seitz pad. The mycelium is Washed on the funnel with about 400 ml. of water to give a total volume of filtrate of about 4.1 liters.

(b) Extracti0n.4 l. of the culture filtrate (1 g. of Compound S) is extracted with four 1 1. portions of chloroform. Evaporation of the chloroform in vacuo furnishes about 596 mg. of crystalline residue. Trituration of this residue with acetone produces about 460 mg. of crystalline material which on recrystallization from ethanol yields about 320 mg. of pure A -pregnadiene-17a,2l-diol-3,20-di'one (1,2-dehydro Compound S), having the following properties: M. P. about 240- 242 C.; [a] |57 (c., 0.70 in chloroform); A31 244 m (e=16,800); \,i?3.003.06;/. (OH),

5.81,: (20-keto), 6.00 6.18M, 6.2351. (A -3-ketone) Analysis.Calcd. for C H O (344.43): C, 73.22; H, 8.19. Found: C, 73.14; H, 8.08.

The substance is indistinguishable from an authentic sample of 1,2-dehydro Compound S.

Example 2 Step (a) of Example 1 is repeated substituting Compound S 2l-acetate for Compound S. Upon extraction of the culture filtrate in accordance with the method of step (b) of Example 1 A -pregnadiene-17a,21-diol-3,20-dione is obtained which is indistinguishable from the product formed in Example 1 or an authentic sample.

3, Example 3 (a) Fermentatin.A medium of the following composition is prepared: cornsteep liquor solids, 3.0 g.; NH H PO 3.0 g.; CaCO 2.5 g.; soybean oil, 2.2 g.;

Compound S, 0.5 g.; and distilled water to make 1 liter.

The medium is adjusted to pH 7.0 -0.1. Then, 100 ml. portions of the medium are distributed in 500 ml. Erlenmeyer flasks, and the flasks plugged with cotton and sterilized in the usual manner (i. e. by autoclaving for 30 minutes at 120 C.). When cool, each of the flasks is inoculated with 5-10% vegetative inoculum of Cylindrocarpon radicicola ATCC 11011 [the vegetative inoculum being grown from stock cultures (lyophilized vial or agar slant).for 48-72 hours in a medium of the following composition cornsteep liquor solids, g.; brown sugar, 10 g.; NANO 6 g.; ZnSO 0.001 g.; KH PO 1.5 g.; MgSO -7H O, 0.5 g.; CaCO 5 g.; lard oil, 2 g.; distilled waterto. make 1 liter]. The flasks are then placed on a reciprocating shaker (120 one and one-half inch cycles per-minute) and mechanically shaken at C. for 3 days. The contents of the flasks are then cooled and, after the pH of the culture is adjusted to about 4:02 with sulfuric acid, filtered through Seitz filter pads to separate the mycelium from the fermented medium.

(b) Extracti0n.The culture filtrate (4 liters) obtained in (a) is extracted with 4 l-liter portions of chloroform and the resulting chloroform extract is evaporated to dryness in vacuo. The residue (about 1.5 g.) is dissolved 'in benzene, and hexane is added to incipient turbidity.. The resulting solution is chromatographed on g. of silica gel. The early eluates yield A -testololactone,-M. P. about 216218 having an infrared spectrum identical with that of an authentic sample. Subsequent elution with chloroform furnishes as the major product A -pregnadiene-17a,21-diol-3,20dione, M. P.

about 242-244; [041 +57 (chloroform).

: Example 4 from the above crystallization are evaporated to dryness in vacuo (about 320 mg.), dissolved in 3 ml. of chloroform and 15 ml. of benzene and chromatographed on 7 g. of silica gel. Crystalline material (about 59 mg.) is eluted with chloroform (1650 ml.), which is identified as A 'i -pregnadiene-l7u,21-diol-3,20-dione by its melting point (about 242-244) and infrared spectrum.

Continued elution with 5% acetone in chloroform (300 ml.) and 100% acetone in chloroform (700 ml.) furnishes a second component, which after crystallization from acetone has the following properties: M. P. about 193-194"; [a] +27 (c., 0.40 in chloroform);

R231; 245 my. (=17,100); k111i? 2.85;.4, 3.00;; (OH), 6.01m 6.21 6.26 (A -3-ketcne) Analysis.Calculated for C H O (346.45): C, 72.80; H, 8.73. Found: C, 72.66; H, 8.58. 'The above-cited properties indicate that this substance represents A -pregnadiene-17a,20fl,21-triol-3-one.

(c) Acylation of A -pregnadiene-17a,20;8,21-tri0l-3- one.-38 mg. of A -pregnadiene-17a,20 8,21-triol-3-one is acetylated with 1 ml. of pyridine and 1 ml. of acetic an- 4 hydride. The resulting diacetate has the following properties: M. P. about 174-176"; [a] +101 (c., 0.43 in chloroform) 8313-, 244 m (6: 17,200) Y; mg 2.85 (OH), 5.75 1 (acetyl), 6.02;, 6.15 6.23 (A -3-keto) Analysis.Calculated for C H O (430.52): C, 67.74; H,-7.96. Found: C, 69.49; H, 7.72.

A -pregnadiene-17u,20;3,21-triol-3-one can be converted into A -pregnadiene-17a,21-diol-3,20-dione 21- acetate by acetylation with one mole of acetic anhydride to form the 21-monoacetate, followed by oxidation with chromic acid in acetic acid. The 21-acetyl group can then be removed by hydrolysis with potassium carbonate in methanol to give 1,2-dehydro S.

In a similar manner A -pregnadiene-17a,20;3,21-triol-3- one can be monoand/or diacylated using ether acid anhydrides or acyl halides.

The invention may be otherwise variously embodied within the scope of the appended claims.

We claim:

1. A process which comprises subjecting a steroid selected from the group consisting of A*-pregnene-17a,21- diol-3,20- dione and 21-esters thereof to the action of the enzymes of fungi selected from the genera consisting of Cylindrocarpon and Fusarium in an aqueous medium containing assimilable sources of carbon and nitrogen, and recovering the 1,2-dehydrosteroid formed from the medium.

2. The process of claim 1 wherein the fungus is Cylindrocarpon radicicola.

3. The processof claim 1 wherein the fungus is Fusarium javanicum var. ensiforme.

4. The process of claim 1 wherein the 1,2-dehydro steroid recovered is' A regnadiene-17a,21-diol-3,20- dione. V

5. The process of claim 3 wherein the 1,2-dehydro steroid recovered is A -pregnadiene-17a,20fi,21-triol-3- one.

6. The process of claim 5 which includes the additional steps of monoacylating the A -pregnadiene-17a, 20 3,21-triol-3-one and-oxidizing the 21-acyl derivative thus formed to a 21-acylate of A -pregnadiene-l7u,2l-diol- 3,20-dione.

7. The process of claim 1 wherein the steroid is A- pregnene-17a,21diol-3,20-dione.

8. A steroid selected from the group consisting of A pregnadiene-17a,20;8,21-triol-3-one and esters thereof.

References Cited in the file of this patent UNITED STATES PATENTS OTHER REFERENCES Bessey: Morphology and Taxonomy of Fungi, 1950, The Blakiston Co., Phila., pp. 15-18.

Vischer et al.: Experientia, vol. IX, No. 10, 1953, pp. 371-372.

Ainswarth et al.: A Dictionary of the Fungi, 4th ed., 1954, The Commonwealth Mycological Institute, Kew, Surrey, pp. 432-433. 

1. A PROCESS WHICH COMPRISES SUBJECTING A STEROID SELECTED FROM THE GROUP CONSISTING OF $4 PREGNENE-17A,21 DIOL-3,20-DIONE AND 21-ESTERS THEREOF TO THE ACTION OF THE ENZYMES OF FUNGI SELECTED FROM THE GENERA CONSISTING OF CYLINDROCARPON AND FUSARIUM IN AN AQUEOUS MEDIUM CONTAINING ASSIMILABLE SOURCES OF CARBON AND NITROGEN, AND RECOVERING THE 1,2-DEHYDROSTEROID FORMED FROM THE MEDIUM. 